Endogenous opioids regulate not only the body's response to pain and stress but also more complex ?unctions including affect, mood, drive, and reinforcement. Control of opioid signalling occurs primarily at the level of release, precursor processing, and synthesis. Transcriptional control of opioid peptide synthesis is an important aspect of opioid signalling and may contribute to the mechanisms of narcotic tolerance, dependence, and withdrawal as well as the pathology of mood, anxiety, and other psychiatric and neurological disorders. In more general terms, an understanding of the molecular mechanisms regulating gene expression of the opioid precursor, proenkephalin, will not only improve our understanding of the processes listed above, but will also contribute to an understanding of the mechanisms regulating the synthesis of other key neuronal signalling components, including neurotransmitter receptors and ion channels. Trans-synaptic regulation of proenkephalin gene expression is an ideal model system to examine the molecular mechanisms controlling neuronal signalling plasticity because multiple forms of trans-synaptic regulation have been demonstrated in a variety of model systems and because the gene and its regulatory regions are well characterized. The primary objective of my proposed research is to determine the molecular mechanisms regulating expression of the opioid peptide precursor gene, proenkephalin, in response to synaptic inputs. Specifically, I propose an in depth analysis of the DNA binding proteins which mediate trans-synaptic activation of transcription of the proenkephalin gene via their interaction with an already identified cAMP and phorbol ester inducible DNA enhancer, as well as attempts to identify and characterize other functionally important DNA elements. In the period covered by this proposal, studies will focus on achieving a molecular description of the functional interactions between synaptic signals transduced by intracellular second messenger pathways, the DNA enhancer binding which mediate synaptic signals, and the individual DNA elements comprising the inducible enhancer. Ultimately, all the essential and modulatory components of the inducible enhancer complex will be identified and thoroughly characterized, such that the system can be functionally reconstituted, and the mechanisms of second messenger and protein kinase inducible enhancer function investigated. The proposed research is clearly of a basic nature and there is no doubt that it will be of fundamental significance for an understanding of the mechanisms regulating nerve cell signalling, opioid physiology, long-term information storage, cell growth, and differentiation.